An improved purification method for cytosolic malate dehydrogenase from several sources

A new purification method for cytosolic malate dehydrogenases from several sources has been developed. The procedure, employing chromatographies on 5'AMP-Sepharose, DEAE-Sephacel and Blue-Sepharose, allows for a rapid isolation of the enzyme (approximately 40 hours), in large quantities, with good yields (45-54%). The specific activity of final preparations were around 1300 I.U./mg and were judged homogeneous by polyacrylamide gradient gel and sodium dodecyl sulfate polyacrylamide gel electrophoresis, high performance size exclusion chromatography and isoelectric focusing.     

Direct microsensor measurement of nitric oxide production by the osteoclast.

Nitric oxide (NO) triggers marked osteoclast retraction which closely resembles that due to Ca2+. The effect of Ca2+ has been attributed to a stimulated release of NO. Here, we show for the first time, by direct measurement with a microsensor, that osteoclasts do indeed produce NO and that this production is enhanced by a high Ca2+. We also show that the Ca2+ ionophore, A23187, mimics the latter. Furthermore, osteoclasts on dentine produce more NO than osteoclasts on glass and NO release from dentine-plated osteoclasts is much less sensitive to stimulation by Ca2+. Finally, the microsomal Ca2+ store-depleting agent, thapsigargin, attenuates NO release only from osteoclasts on glass, suggesting that stored Ca2+ has the dominant effect in modulating NO release from non-resorbing cells. NO is a powerful inhibitor of bone resorption: a direct demonstration of its production is therefore strong evidence for a role in modulating osteoclast function.     

Phenotypic characterization of ouabain-resistant Aedes albopictus cells.

The phenotype of a ouabain-resistant Aedes albopictus cell line has been partially characterized. Treatment of ouabain-sensitive cells with 0.005-1.0 mM ouabain resulted in an 80% reduction in the uptake of 86rubidium (86Rb+), an ion with an affinity for the K+ pump binding site; ouabain-resistant cells showed only a 40% reduction with 1.0 mM ouabain. When ouabain-sensitive cells were incubated in the presence of ouabain (0.1 mM) for one and one-half to three hours, the molar ratio of intracellular Na+/K+ rose from 0.2 to 4.2. In ouabain-resistant cells, a similar treatment had very little effect. Based on [3H] ouabain-binding studies, ouabain-resistant cells were estimated to have 60% fewer binding sites per cell than ouabain-sensitive cells. The spontaneous mutation rate from ouabain sensitivity to ouabain resistance was calculated to be 1-6 x 10(-8) mutations/cell/generation, a value similar to that reported for mammalian cells at the analogous locus.     

Effect of a low-frequency magnetic field on systemic arterial pressure in spontaneously hypertensive rats

The effect of low-frequency magnetic field (MF) on systemic blood pressure has been studied in chronic experiments on 21 spontaneously hypertensive rats. The animals' kidney area was exposed to MF (induction value 30T). Direct blood pressure measurements have revealed an antihypertensive effect.     

Characterization of a substance P-Gly12 amidating enzyme in human cerebrospinal fluid

Enzyme activity capable of converting the glycine-extended substance P precursor, substance P-Gly12, into substance P was purified from human cerebrospinal fluid. The conversion reaction was monitored by radioimmunoassay measurement of substance P formation. The chemical identity of the product was verified by reversed-phase HPLC. The enzyme reaction was stimulated by Cu(II) ion and ascorbic acid and inhibited by the presence of diethyldithiocarbamate. By HPLC molecular sieving, the major enzyme activity appeared as a protein of 26,000 molecular weight.     

Differetial sensitivity of spontaneously hypertensive rats to the hypothermic effect of dopaminergic drugs

The dopaminergic agonist apomorphine produces dose-related hypothermia in naive rats as does L-DOPA in carbidopa-pretreated rats. The hypothermic responses to these two dopaminergic drugs were significantly more pronounced and prolonged in the spontaneously hypertensive rat than in normotensive Wistar control rats. The greater sensitivity of the SHR to these drugs was reflected as a leftward shift of the dose-response curves for apomorphine- and L-DOPA-induced hypothermias.